Project 2: Abstract Antibody formation against coagulation factor VIII (FVIII) is a major and serious complication in current protein replacement therapy for the X-linked bleeding disorder hemophilia A. Approximately 20-30% of patients develop neutralizing antibodies (?inhibitors?) that inhibit coagulation activity, thereby complicating treatment, increasing risks of morbidity and mortality, and raising treatment costs. FVIII can elicit very high-titer antibody formation despite being given intravenously at low antigen doses. FVIII-specific B cell responses are dependent on CD4+ T helper cells and require co-stimulation. However, surprisingly little is known about the in vivo mechanism of FVIII antigen presentation to T cells or B cell activation. In this proposal, we seek to answer which antigen presenting cells (APCs) are required for MHC II presentation to CD4+ T cells (leading to FVIII- specific B cell activation), how these APCs interact to prime FVIII-specific CD4+ T cells, which subsets of CD4+ T cells are induced to promote B cell activation (including T follicular helper, Tfh, cells), and how innate immune signaling may alter these events. Working with the other projects, we will use these newly established experimental models to determine the impact of signals derived from the microbiome and the effect of altered molecular structure of FVIII. We propose three specific aims. Aim 1 is to define the mechanism of in vivo MHC II presentation of FVIII antigen to CD4+ T cells. We will utilize fluorescently labeled FVIII to study in vivo antigen uptake, and establish an in vivo MHC II presentation assay based on epitope-tagged FVIII. We will use a combination of depletion of specific APCs, confocal microscopy, and intravital 2-photon microscopy to determine the requirements and roles of the critical APCs, their location in lymphoid organs, and their interactions. We will visualize dendritic cell and T cell migration and clustering of specific T cells around APCs, and interrogate innate and cytokine responses that increase B cell activation. Aim 2 is to define the mechanism of in vivo activation of FVIII-specific CD4+ T cells and B cells. Using a combination of depletion experiments/knock-out strains, adoptive T cell transfer studies, flow cytometry, and cytokine assays, we will determine the requirements for T and B cell activation, and the subsets of CD4+ T cells that activate B cells and their requirements for co-stimulation (with particular emphasis on the role of Tfh cells in primary and memory B cell activation). Aim 3 is to determine the effects of signals derived from innate immune sensing and the microbiome on B and T cell activation against FVIII. Here, we will study the effects of TLR9 agonists and anti-inflammatory drugs on dendritic cell subsets as an example of a how inflammatory signals alter inhibitor formation. In collaboration with Project 3, we will determine how an immune stimulatory or suppressive microbiome alters FVIII antigen presentation, CD4+ T cell activation, and Tfh responses.